An estimated 150 million people world-wide are infected with the hepatitis C virus (HCV), a major cause of chronic liver disease. The nonstructural protein 3 (NS3) of HCV is a multi-functional enzyme in the replication cycle of the virus and requires NS4A as cofactor for proteolytic activity. Inhibition of the NS3/4A protease activity by HCV-specific antiviral compounds is presently regarded as a promising strategy for coping with this disease. Despite continuing efforts, the development of effective NS3/4A protease inhibitors has been only moderately successful to date. We propose a novel strategy for the development of inhibitors, which takes advantage of the proximity of the NS3 active site to the NS4A cofactor- binding site. Two different combinatorial approaches will be used to identity (a) active site-directed small molecule inhibitors of the proteolytic activity of NS3/4A and (b) small molecules which disrupt formation of the NS3:NS4A complex. These two types of inhibitors will be coupled in all possible binary combinations using linkers of varying length. This library of linked inhibitors will then be tested to assess whether the expected cooperative binding enhances the affinity and effectiveness of the linked inhibitor on protease activity and cofactor binding as compared to the unlinked parent inhibitors. PROPOSED COMMERCIAL APPLICATION: Hepatitis C virus (HCV) has infected 3.3 percent of the world's population. In 1997, it was estimated that nearly four million people in the United States have been infected with HCV, about 30,000 new cases are expected to be diagnosed each year. New therapeutic drugs, potentially in combination with interferon therapy, are urgently needed to provide safe and effective means of HCV treatment. Market analysis estimates that the value of such drugs has potential for producing more than a billion-dollar per year income.